Copyright © 2005 by the Genetics Society of America DOI: 10.1534/genetics.l05.042093 Segmental Structure of the Brassica napus Genome Based on Comparative Analysis With Arabidopsis thaliana Isobel A. P. Parkin,*1 Sigrun M. Gulden,* Andrew G. Sharpe,* Lewis Lukens,Ť Martin Trick,1 Thomas C. Osborn§ and Derek J. Lydiate* * Saskatoon Research Centre, Agriculture and Agri-Food Canada, Saskatoon, Saskatchewan S7N 0X2, Canada, University of Guelph, Guelph, Ontario NIG 2W1, Canada, *John Innes Centre, Norwich NR4 7UH, United Kingdom and ^Department of Agronomy, University of Wisconsin, Madison, Wisconsin 53 706 Manuscript received February 15, 2005 Accepted for publication July 11, 2005 ABSTRACT Over 1000 genetically linked RFLP loci in Brassica napus were mapped to homologous positions in the Arabidopsis genome on the basis of sequence similarity. Blocks of genetically linked loci in B. napus frequently corresponded to physically linked markers in Arabidopsis. This comparative analysis allowed the identification of a minimum of 21 conserved genomic units within the Arabidopsis genome, which can be duplicated and rearranged to generate the present-day B. napus genome. The conserved regions extended over lengths as great as 50 cM in the B. napus genetic map, equivalent to ~9 Mb of contiguous sequence in the Arabidopsis genome. There was also evidence for conservation of chromosome landmarks, particularly centromeric regions, between the two species. The observed segmental structure of the Brassica genome strongly suggests that the extant Brassica diploid species evolved from a hexaploid ancestor. The comparative map assists in exploiting the Arabidopsis genomic sequence for marker and candidate gene identification within the larger, intractable genomes of the Brassica polyploids. ARABIDOPSIS thaliana (hereafter referred to as Arabidopsis) is one of almost 3500 species that make up the monophyletic family of the Brassicaceae (Price et al. 1994). Arabidopsis thus shares recent common ancestry with a large number of species of significant economic importance, including a diverse range of vegetable and oil producing crops, the majority of which are Brassica species. Arabidopsis is an excellent model system for the Brassicaceae, with a small and relatively simple genome, efficient transformation system, diverse range of genetic and genomic resources, and a completed genome sequence (Arabidopsis Genome Initiative 2000). Over the past 10 years, plant comparative mapping has taken prominence as a powerful tool, first, for uncovering the processes and rate of genome evolution and, second, for allowing the transfer of genetic resources between species. Comparative mapping has been most extensively applied to the grasses where the genetic maps of 11 species, including the model mono-cot rice, have been aligned. These include 11 diverse species varying dramatically in haploid chromosome number, genome size, and phenotype (reviewed in Sequence data from this article have been deposited with the EMBL/ GenBank Data Libraries under accession nos. CZ692804—CZ692942 and DR697805-DR697867. 1 Corresponding author: Saskatoon Research Centre, Agriculture and Agri-Food Canada, 107 Science Place, Saskatoon, SKS7N 0X2, Canada. E-mail: parkini@agr.gc.ca Devos and Gale 2000). Perhaps the most striking observation from the cereal studies was the extensive genome conservation observed between species that diverged millions of years ago. Using rice as the basal genome, <30 conserved blocks were identified, which could be rearranged and/or duplicated to form each of the other grass genomes. Comparative mapping studies among members of the Brassicaceae have been more ambiguous in their conclusions, leading to ongoing discussions about the level of genome duplication prevalent in modern Brassica cultivars and the extent of the genome rearrangements that have occurred in the evolution of these cultivars from a common ancestor (Lagercrantz 1998; Lan et al. 2000; Lukens et al. 2003). This study focuses on the genome of the oilseed crop Brassica napus, which is an amphidiploid species formed from multiple independent fusion events between ancestors of the diploids B. rapa (A genome donor) and B. oleracea (C genome donor) (U 1935; Palmer et al. 1983; Parkin et al. 1995). Polyploidy is a prevalent evolutionary mechanism within angiosperms since it has been estimated that 30-70% of modern plant species have evolved through a polyploid ancestor (reviewed in Wendel 2000). Polyploidy can occur through either the duplication of whole-chromosome complements or the fusion of related chromosome complements, and stabilization of the newly expanded karyotype must then take place to ensure normal diploid inheritance. Diploidization of the novel polyploid can occur through Genetics 171: 765-781 (October 2005) 766 I. A. P. Parkin et al. chromosomal restructuring or genetic control of illegitimate recombination events or a combination of both mechanisms. It is widely accepted that the progenitor diploid genomes of B. napus are ancient polyploids and that large-scale chromosome rearrangements have occurred since their evolution from a lower-chromosome-number progenitor (Schmidt et al. 2001). What is more contentious is whether the diploids evolved through a hexaploid ancestor or whether they were formed via segmental duplication of one or two ancestral genomes (Lukens et al. 2004). B. napus, a relatively young amphidiploid, is somewhat of an anomaly since it has been established that no major chromosomal rearrangements have occurred since the fusion of the progenitor A and C genomes, but homeologous recombination events between these two related genomes are common in newly resynthesized B. napus lines and have been observed at low levels in established canola cultivars (Parkin et al. 1995; Sharpe et al. 1995; Udall et al. 2004). It has yet to be established if B. napus has evolved or inherited a locus controlling homologous pairing similar to the Phi locus in hexaploid wheat ( Jenczewski et al. 2003). Comparative mapping between B. napus and Arabi-dopsis has thus far targeted small regions of the Arabi-dopsis genome, generally identifying three colinear segments in each of the diploid genomes for every region of Arabidopsis studied, thereby promoting the idea that the diploid Brassica species may have evolved through a hexaploid ancestor (Osborn et al. 1997; Cavell et al. 1998; Parkin et al. 2002). However, at the same time regions suggesting a more complex relationship between the two species were also identified (Osborn et al. 1997; Parkin et al. 2002). In the earliest published global comparison between one of the diploid Brassicas, B. nigra (black mustard), and Arabidopsis, an extensive number of rearrangements were invoked to explain how the two extant diploid genomes evolved from a common hexaploid ancestor (Lagercrantz 1998). There have been four global comparisons of the genomes of B. oleraceaand Arabidopsis. Although all have been limited by a low density of common loci, three identified extensive synteny between the two genomes but were inconclusive in assessing the level of duplication of the colinear segments (Lan et al. 2000; Babula et al. 2003; Lukens et al. 2003). A more recent comparison of the B. oleracea and Arabidopsis genomes refuted the possibility of a hexaploid ancestor, citing evidence of colinear blocks ranging in copy number from 1 to 7 (Li et al. 2003). This study describes a comprehensive comparison of a Brassica genome with that of Arabidopsis. Sequences of 359 probes derived from Brassica and Arabidopsis that detect 1232 genetically mapped loci in B. napus were used to query the Arabidopsis genome, revealing 550 homologous sequences and their inferred chromosomal positions. The data provide strong evidence to support the hypothesis that the Brassica diploid genomes evolved through a hexaploid ancestor and suggest conservation of some centromeric regions between the two species. The postulated ancestor appears to have been formed from duplication events that occurred subsequent to the putative global duplication events that took place between 65 and 90 million years ago (MYA) during the evolution of Arabidopsis (Lynch and Conery 2000; Simillion et al. 2002; Raes et al. 2003). The resultant genetic and physical comparative map can be used not only to infer genome rearrangements during the evolution of the Brassica species but also to identify regions of the Arabidopsis genome that may harbor genes of interest and should potentiate the exploitation of Arabidopsis genomic tools in Brassica research. MATERIALS AND METHODS Genetic linkage analysis: Genetic linkage analysis in B. napus was carried out as described previously except hybridizations with Arabidopsis clones were washed only at low stringency (2X SSC, 0.1% SDS) (Sharpe et al. 1995). The B. napus population consisted of 60 doubled haploid lines derived from crosses between a winter B. napus breeding line (CPB87/5) and a newly resynthesized B. napus line (SYNI) as described in Parkin et al. (1995). The genetic map also includes loci positioned through previously described map alignments with a second linkage map of B. napus and one of B. oleracea (Bohuon et al. 1996; Parkin and Lydiate 1997). Briefly, common parental genotypes allowed corresponding loci to be identified between the maps through the inheritance of identical restriction fragment length polymorphism (RFLP) alleles. Loci mapped in only one population that cosegregated with such common loci were positioned at that locus in the combined map. Loci mapped in only one population positioned between common loci were placed in the corresponding interval in the combined map on the basis of their relative position in the map of origin. The RFLP probes consisted of 213 Brassica genomic clones (pN, pO, pR, pW: Sharpe et al. 1995), 61 BrassicacDNAclones (CA, es), 88 Arabidopsis cDNA clones (I, N, R, Z: Sillito et al. 2000), and 6 cloned Brassica or Arabidopsis genes (ACYL, CONSTANTS, FCA, HS1, oleosin: pC2, A9 desaturase: pC3). The genetic linkage map was constructed using Mapmaker v3 with a LOD score of 4.0 (Lander et al. 1987) and the linkage groups were drawn using Mapchart (VoORRlPS 2002). Irregularities in meiotic pairing in the resynthesized B. napus parental line of the doubled haploid population used for the initial and the additional mapping caused a nondisjunction event that prevented the accurate mapping of further loci to linkage group N16 (Parkin et al. 1995). A limited map of N16 derived from the alignment of Nl 6 from B. napus, described in Sharpe et al. (1995) and 06 from B. oleracea, described in Bohuon et al. (1996), has been used in the present analysis. A similar alignment of Nl 6 and 06 was discussed in Ryder et al. (2001). Sequence analysis: Brassica genomic or cDNA clones were sequenced from each end using the BigDye v2 Terminator cycle sequencing kit according to the instructions of the manufacturer and subsequently the reactions were run out on an automated ABI377 DNA Sequencer (Applied Biosystems, Foster City, CA). The Brassica sequences were analyzed using Sequencher (Gene Codes, Ann Arbor, MI) to trim vector sequence, identify overlaps, and generate contigs. Brassica and Arabidopsis/5. napus Comparative Map 767 Arabidopsis sequences were analyzed for homology to the The Institute for Genomic Research Arabidopsis pseudochromo-some genomic sequence version 5.0 (ftpi^ftp.tigr.org/pub/ data/a_thaliana/) using the BLAST programs of the National Center for Biotechnology Information (http://www.ncbi.nlm. nih.gov/) housed on a Linux server. Low-complexity sequences were filtered in the BLAST analysis, and default values for cost (mismatch cost, —3.0), reward (match reward, 1.0), and word size (11 bp) were selected. The default gap opening penally (5.0) and the gap extension penally (2.0) were also selected. Perl script was used to extract the base-pair position in the Arabidopsis genomic sequence of each high-scoring segment pair (HSP), identified with BLASTN, for each clone where the primary HSP had an £-value of 55.2- 55.9' 70.6- 90.9-93.1- 97.6" 99.3- 101.0- 104.9- l\ /~N -iBt2r JpW157bCA36a IpNIBÖC JCAIOOb pW239b 1CA53bpW145a -P0173E1 Jp043E1 ICO&J 1CAÄTV3 /lpN206b_72jrB2a /■ p052b - mi330c HlD11bes2659c ^pN107cpW10SE1 N96493e /■CA142e ^■pW179c /pC2b Jp017a pR36a IG12b l-USK'3 IC01cpN97d PN152E1 pOllBEl pN47E3 pW120b pW101d CA138V2 T14233d mil 37b pN173d_72 pO70e pR36fpN53f pN129a_72 pCMSBE' mi138f pNiSOn pW108c CA73aCA1t9b JIA026 IC01RI7 lM65549b -pW201c HT44979C CA144b -[IN121E2 -CA12CS -p012c -pW136c HpW172bpN1Ji. -pR85b N-^ 70.9 13' J- \y ^ -pOUTb -CA120* ■ CAUW ■ mi97a na )mi174cpNt2lH ■pR64a ICÍÍ+ÍBATTS25MC ICAAa pNiCao -EOii pN91U pM1»m iC04d IG01S IBüSa IHDEb mi138h ptfnMb CA39b p*T2*a pR30a_72 IGD4P IA09Í pNSifc pR115a (OS7c p085c /Ci0eiAV154» lCA117b pW161a pW150d p085g p086a_72 CA76f p03b IA04a pW2Q7a ID02b CA37d pW180b pR4bpN181a IH02a Z1779aa T4J9SSh CA111C pViHWa JCUId ATTS2C*1b ea 1732b IBCMa pR72a orrp5a mi271c IBÖ6RI7 mi125b pWi78tj pOlľaa CATIcCAS» mi219b |jQ119a 109.6—W— |W191« Figure 1.—Genetic linkage map of 5. napus. Linkage groups are arranged according to the regions of primary homology between the A (N1-N10) and C (N11-N19) genomes (Parkin et al. 2003), with centimorgan distances indicated to the left of each group. Each genetic locus is colored according to the presumed Arabidopsis homolog: light blue, chromosome 1; orange, chromosome 2; dark blue, chromosome 3; green, chromosome 4; and red, chromosome 5. Loci in italics were found within conserved blocks on the basis of secondary or tertiary hits within the Arabidopsis genome. Loci duplicated within a B. napus linkage group are indicated by vertical lines to the right of the group. Identified genome blocks showing conservation of marker content and marker order between the Arabidopsis and B. napus genomes are shown to the left of each linkage group. Each block is colored and labeled according to the identified homologous region in Arabidopsis (see Figure 2). Inversions identified in Brassica relative to Arabidopsis are indicated by arrows. Regions of the B. napus genome that have been tentatively aligned with Arabidopsis centromeric regions are indicated by hatched blocks. Identification of conserved blocks between Arabidopsis and B. napus: For each B. napus linkage group it was possible to identify blocks of conserved synteny between B. napus and Arabidopsis, which represent chromosomal segments that have been maintained since the divergence of Arabidopsis and Brassica from a common ancestor (Figures 1 and 2). A conserved block is defined as a region that contains several closely linked homologous loci in both the Arabidopsis and Brassica genomes. Each block has a minimum of four mapped loci with at least one shared locus every 5 cM in B. napus and at least one shared locus every 1 Mb in Arabidopsis. Using these criteria, each conserved block contained on average 7.8 shared loci and Arabidopsis/5. napus Comparative Map 769 N12 N3 N13 -icA-i2nrpwii6Ei ' r'. ■■' ' ■ i::.l ■CAT1Í pM3E2 pí*121q CA4C phHQří E05hCOd pNS1eCAí(DC pNfSW cMMEl P0136E1 ltŕ*a IGDIblBK« ihmľ nasí9i: pRS6E1 (AV102Q mi90c CA39e IA04RV9 IA10h pRÍSb .;■-. pf-*33b pRH5c POÍ7EÍ H5SS133 lGU7d pFT34EÍ p085a pWlÄ4h IG02C /CÍ0fT41629b CA117a pW161b pNfO&S IA04b pW207b ID02C CA37c pWiaOc pR4a IH02b es4424b pW148E2pW177E3 p'.vaiuí itwat p>V13EtŕZ1T7gm. I IB01RI3pO12SE2 I pR72b .'OH9r l p05b_72 |pR20E1 P/J1E7E1 IPIÍJES poiaut J ICATřVM pWHIĚI ' pWt9lb 48.7' 78.E CA12fla pKH H3EJZ1d Z33873d ttíSaSSb pWlSJj :: a .' ■ :i CA156b pN121b 4JRE4g CA1JDV1 SJW153P IEŮÍIptti02c_7J (jrt'169a pOtBOo p*ťl06a pNIBOb IBOHí IG01C RaMBfl(l rwiräí IA10] -iGíi4dpir:2i4^ :.'.V1-I-I> IAŮ9, -QTä&n pW102ĽH36913e CA1J1b DOSi IC12a CASBf M2M -PW133S 72 ŕBU« CA2bpNl*7d irOr. N95848a es4671b'jA"ib F201D8C pNlfľe pN213g pW143a CA55C IH02e ID01I 2Zů iCAUTc ] l2U8:ý)clA101 J pN99d 4lA04ei'.:uer JpjN1B7břNÍ4i ~lpNi5ic - pW239c S pN59f -l T439fcí)á CA58I HpOMii pNfifid HpW139b IC12HV2 - I CA58d 1tj56J5i. -pHffQ - FWRH - loořii 77.5—y----CA30V2 Figure 1.—Continued. I. A. P. Parkin et al N14 -pW177E1 -pŕf13ľ \p>-l'}7t_ 71 pMf7Hb 1lhUt3 H 53456 |:-,:'.JipR94E2 1i:€3h 60.1 ' 63,4 \ 70.1 ' 75-V 107 3 /*N pW^SEi f>0147d fjwia pN95a i I I S>25Wb pN53i pW137a ClSc IB08C (■layeiipNbJti : --. \\". IGMf CA58b APSa pN151t>_7Z phH*b 72 '■1- ■■■ pN173b IB08B4es1153a pW108f pwziac pO145E2pO106E1 p09c PW120E2 PW136E2 PW206E2 PW188E2 HSIa^Nip;; IH1Da pW130a_72 p087a pMSflEl T14233b r*Jlpři IF01clG02f pR54c/IA10d ICOTc IA04c pMIBTa 72ph*4*e pN151d" pN59c Maubib CA58i-09ab pN66cpWt39a CA58e - |-.-.1r , pOdp0126E3 PWT43E1 FQSb i lit 17.0- 33.7- 50.5\ 104.5- -pMn-? -pHKH.l IFD5C HCAliapNUMb 72 - pN99c___72 -pC171D_72 - pN95b_72 -pN1Mb J pW 137b E f 2= 1 mi330b IB08d r4.,!,r..,.. pNfiSvi ÍG04I it ľ a CA58a APSb ACYLa TTSŮ62a BSjMSb M65549d T04362c pW217a Z30800C IBOBf p046b es4619c T 13648a pWl15ees4671f CA73a pR114b pW114c (H0e/pN148c pN53d IA02C F20taS* 1 N96307a ■ IC01e 1 pN215a 1 CA144d -p0123c HCA67a es1847f JpN113cpN2d_72 1pW172e - lEObb skeleton of the B. napus genome (Figures 1 and 2). Although coverage of the two genomes is extensive, there are areas where marker density is limited, specifically the regions spanning the Arabidopsis centromeres (Figure 2). The low-copy-number sequences utilized in the Brassica mapping would be expected to have lower levels of similarity to centromeres, since the centromeres tend to be located within gene-poor transposable-element-rich regions (Arabidopsis Genome Initiative 2000). Comparative genome organization: The organization of the B. napus genome in comparison to the Arabidopsis genome as depicted in Figures 1 and 2 has been summarized for each of the linkage groups. Due to the close homology between the A (N1-N10) and C Arabidopsis/5. napus Comparative Map 771 N15 N6 16.9- 41.9 ■> \.A 1211 - -pN21E2 Jes2060d pN23E3 1p092E1 HT04135b IG04h pN5Za p0152E1 l*irng pO105£1 pW224a_72 PW197E1 Z17993C CA149c CA42C - l-iai- PN123E2 pW123E1 pW145d CA25C T22090a T46145a pN216e ID08f PW164E1 p0159bpW138E1 CA390 CA1fi5*rJ49lĚ2 p0143a P0131E1 CA42e es5209a !G09c pN47a 1G10clA01d CA101e ACYL c p013SE2T75Ů62il es4619b pR54e es2298b CA73d pW114a IHOBc pN148d P0128E1 - IA02d HpW197dN96307b JpN215cpOfS3Et lCA'44= 4pN59E2[jN9H -lp0123aCA67b JpOIZdpN 1pN2c 113a "1 iN113b J Hes1847dpN113b -pW172c - IBOSc 128.4—bi—pRB5c Figure 1.— Continued. B 455- 50.5-53.0- 107.8- 111.1-112.8- -es4619a - p09a lT04135a IG04g ■ pN199a NS2bp0152b ■ pW199a pO105a p0165e ■ pN199b ípNUMe pW224b Z17993b CA149b ' CA42a pN 123X2 es1230g pWUSc CA25e T22090b T46145b pN216f ID08e p0159cpOH3b_72 N970fi7d IB09C es5209b -pOllW pR6b lAÍMh lC10í:pO9d pNIBQd ÍesMgzapW1D2d i CA72S CAHd ípNiŮiapOiŽOD -pWieía ( pW137c_72 iAll!>r »118443(1 es1230c pR43b { IBO1clC01h ■C*129u -*»f7J3c -! JCPtfpft3a -pNĚ*a -p011is I (pato im 2b IpWJISd N17 X0\ / p087E2 . , YT JpO104E1 pWIBrtt es4619g lAÜSfl lA1;: ■ I -'■ ■ pR36g pW108b_72 PR93E1 pW197f pO70c pW162b_72 IG02g ■ IA01elG10d esi230e IG09d CA76ap0131E2 p085b CA42f IBQ9d pR36epÜ1E9C1 pN53h i': !. SiNSEti pR3h pNZDCZ pRtlld <:A1I9b pR43a_72 es1230b p05a ;i9J43a IAŮM pW137e PN216E2 pWlh7bpOlíŮS pN64f2 pNIOlr. CA72Í «lüSJtla 53.1 -1 irOBh CAS9S es1230a . pW104E1 pW174E1 T41662a p029a pN97e p052a PC11DĽ3 ID11fes2659d pN107b CA142d pN181g pW179d pR36c pC2f pW101c pW120a IG12g JCA112ap043c "I pW225a IIFOE* CA36d 1CA115b -I IBÜC T42294a (N11-N19) genomes of B. napus, the primary homeo-logs in B. napus (described in Parkin et al. 2003) are indicated in the comparison. Nl/Nll: These two B. napus linkage groups are homologous along their entire length. The top half of each linkage group shows significant homology to the long arm of Arabidopsis chromosome 4 (block C4B) with one inversion, previously noted in Cavell et al. (1998), disrupting the colinearity between the two genomes. The inversion appears to be specific to Nl/Nll and is not present in the homologous regions of linkage groups N3/N17 and N8/N18 where copies of block C4B were found. The lower half of Nl/Nll is homologous to the top arm of Arabidopsis chromosome 3 (block C3A). This block is also strongly conserved in N5/N15 and N3/N13. In each case, the distal end of the Arabidopsis chromosome corresponds with the terminal end of the linkage groups. At the breakpoint between the two large stretches of colinearity, there are three markers that span the centromere on Arabidopsis C3 and additional markers that do not identify a conserved region. One gross chromosomal rearrangement would 772 I. A. P. Parkin et al. N7 N8 N18 0.0 \ [iWlyij 1.2 A //IG02b A 6.3 \\V /// IF01e Jilt »\% H36813C pcnwfípflnsd pQISÍt pMWHÍft mi138b;UV103* !A09fllG04t RS98«Bt> CA39d ibobí i&Dír loa+c pNiaop pWSIZfl po»* mi219a mi322b ŮN105P PN91P pOlBDO pW1S9b pW10BE1 PWTS5E1 pW195a IED5g COb pN1«r CA4d &A40aCA11Da *TT5252Ja p011162 CAltíh pR64E1 p07b p011Bh ATTSříOef pN47E4 ID051 N9f549jj zsjpsb H3flSS1rrpW239E1 r*3c pWÍOOa CAÍ4Í plAMOSa CA120C 29.01 29.5f 38.5/ 39.3', 48.6'/ SD.3ÍJ 570i 61.87 64.7' 75.5/ N10 1 ID01bpW101a_72 - es2298c HR3IM?4ti pN170a T21447a mi291b pN21b IG12d es2060r pN53b_72 p092b pfl34t> pN23f CA20a CA22c ess40a IBQBf ií307a Z179933 mi138a pN199c PR11Sb p0155d pWJ4í)3 IRO*i pwna? iawc kí-;m:..i- IA10Í CA39a CAMt, iHOSs IBŮSc iGOie ICŮ4b mi219cmi322a mi438a pNiaoh CAHÍb pN1D5cpNeiů p01Ha_7Z pW1BM IED5I COa pN1 2; CA110d li"ť!5rATTS2524Í CAÍJ4Í p07apOtífla CA1HJ CA71Ě N96493J Z33S73» naea2ib pnm pW2ÍBb CA54b pWUSd P0147»fltó147d -esíi47b Figure 1.— Continued. block C5E to lie adjacent to block C5A. This pattern of C5A-C5E is conserved on linkage groups N3/N13 and N10/N19. The same inversion moved blocks C5B and C5D to the bottom of N2/N12. N2/N12 share a region of homology with Arabidopsis CI, block C1E, adjacent to which are five markers that flank the centromere on Arabidopsis C4. Two more small conserved regions were identified on N2/N12, C3B, and C5F. One inversion on Arabidopsis C5 and three insertion/deletion/transloca-tion events represent the least number of rearrangements, which could generate the present organization of N2/N12. 774 I. A. P. Parkin et al. TABLE 1 Number of loci originating from each Arabidopsis chromosome, on the basis of sequence homology, for each B. napus linkage group Linkage group Gl C2 G3 G4 G5 ND Total Nl 4 5 14 29 2 6 60 Nil 3 6 13 27 7 7 63 N2 14 3 3 4 34 9 67 N12 12 4 4 7 41 2 70 N3 9 19 17 24 36 2 107 N13 19 16 25 17 42 11 130 N4 6 14 6 7 7 2 42 N14 13 33 10 6 13 6 81 N5 13 13 16 1 5 5 53 N15 36 3 16 2 7 4 68 N6 29 4 3 5 21 1 63 N17 17 11 9 23 18 4 82 N7 32 9 7 3 3 1 55 N16 17 0 3 0 3 1 24 N8 20 1 1 17 4 3 46 N18 31 7 12 7 8 7 72 N9 27 10 9 12 17 1 76 N19 10 4 5 8 57 7 91 N10 14 2 3 2 40 6 67 Total (%) 326 (26) 164 (13) 176 (13) 201 (16) 365 (30) 85 1317 Expected" 313 209 240 187 281 ND, not determined. " Expected number of loci to originate from each Arabidopsis chromosome on the basis of random distribution of loci across five chromosomes with the following approximate sizes: CI, 30 Mb; C2, 20 Mb (not including the NOR region); C3, 23 Mb; C4, 18 Mb (not including the NOR region); and C5, 27 Mb. N3(N17)/N13: The homology of N3/N13 to C5 is described above, below which N3/N13 share homology with Arabidopsis C2 (block C2BC). Block C2BC on N3/N13 was defined by a lower density of comparative markers, which were further rearranged by an inversion, compared to the duplicated copies of C2BC found on N4/N14 and N5. The lower end of C2BC on N3/N13, which borders the centromere on C2, lies adjacent to a conserved block originating from the centromeric region of Arabidopsis C4 (block C4A). Below C4A, N3/ N13 share homology with block C3A as described above. At the junction of C3A, which lies proximal to the centromere on C3, N3 is no longer homologous to N13 but instead shares homology with linkage group N17 and Arabidopsis C4 as described above. The remainder of linkage group N13 has no clear region of homeology in the B. napus A genome. However, in relation to Arabidopsis, this region of N13 shares homology with the blocks flanking the centromere of C3 (C3B-C3C), block C1B, and block C4B. In the area that would be homologous to the centromeric region of C3, there are eight markers with homology to different Arabidopsis chromosomes, three of which flank the centromere on C2. At least three gross chromosomal rearrangements and two inversions are necessary to generate N3 from the identified conserved blocks; assuming that C3ABC has been essentially conserved, one additional translocation/insertion would be necessary to generate N13. N4/N14/N5: The majority of N4 and N14 (65 and 75% of the mapped length, respectively) and the upper half of N5 share homology with Arabidopsis C2. The organization of N14 suggests that of an isocentric chromosome with the upper and lower arms sharing numerous common markers mapped in inverse orientation with respect to each other. The top of N4 and the homeologous central section of N14 show small blocks of colinearity with Arabidopsis C3, C4, and C5; N14 has one additional block from CI. Three gross chromosomal rearrangements are sufficient to describe the organization of N4 and one additional inversion and two translocation/insertions would describe N14. N5/N15/N6: The lower half of N5 and N15 as described above (for Nl/Nll) are colinear with the long arm of Arabidopsis C3. At the center of N5/N15, the markers originate from Arabidopsis CI, with comparative markers flanking the centromere on CI. This central region on N15 is part of a larger block, which is colinear with the upper arm of Arabidopsis CI and the homeologous region of B. napusNQ. One and two large chromosomal rearrangements would generate the present organization of N15 and N5, respectively. Arabidopsis/5. napus Comparative Map 775 AtC1 AtC2 AtC3 ID01 ■ pW177(2) ■ pN170-CA20 pN21 <■ es2060-pN23 p092ť T04135-1(304(2) -pO152pW199pO105 pOIES p0145pN199 pW224 pN34 Z17993 CA42(2) pN123 pW123 pW145|2) CA25ID08T22090T46145 pN216 pW164 p0159 pW138 p0143 p0131 CA42 poas 635209 CA76(2) IG09 es1230 pW146 pN47J7A IG10 lAO-Hy/' CMOl'//. N95848 'j/ pOS7 pW162IA10IE03 pN96 ACYL p0136 pW23S T04362N65549 es4424(2|i m y ess40 n CA22^ IB0B- pN95- PN168- pW217- Z30B00- IB06 p046r es4619' pR54> pW221 * pW22B- T20808 - pN173- pW122 - pW203pO130K p014S(2)' H36320 ' T42294 J CA137pO10fi<- p09' CA117pN2pW161ř T41629 ' pW150 - pOB6pW134ľ IC10(4) n IG10(2)IA01(2)h CA76pR60r p03- IA04- PO104H77224I- pW207' pR94- IH08 — CA149 — IDD7 CA37IM2>— pO10 — pW180 -*■ A p0142 -es1732- CA129- p0153- J \ -1D >B -15 *\ ^C —20 pN59(2) - CA157-IF0S-. PW186CA87!. pHSzS pW194 v CA86-IC07- C A137(2) R90150 k, IC06N PN167F20108I, CA15\ pN44_pN151 T46379Í- p059CA111«, p098 IC11 Vv pN67 pNBeOi pR64(2| A) = pN22 0A IE03(2)IA10(3|0V — pW139>V — ID05(3)\N — IC12AN_ IB01(3) pW112k pN120pW133r pO170_O171 - CA21, HSI(BLASTX)* pR113v T45845 v pOS5(2)~ pN194CA9t pC3- CA12- pR97- ID01{4! IF0SI- pW143pN174K pN97(2| 's*-) pN13-/W CA16(2)es3665ř pR85 IE05(2) pW15S- pW172- pN113pR85(3)l- CA67- p012- IE05(3) - es2533 - CA120- p0123' ID05(2) - CA144Z26226T44979h p0153(2)pN215K A pW142- pW181 pW201 f es1847 N96307 ( IA02- p0128- pN148- IH08(2) - >B \ Y J ^D pN53- IC10(2)- es229B(3) - p0172- IC10(3) - pR6 T45966 pW191(2) R30025 pN59 pW104~: pW174 ID05 pN99 T20671 IA09(4) pW130 CA103 pWÍSS IH10 CA2(2) HS1(2:BLASTX) pW205 pW101j>W120 IDQ1|5) CA16 A >C Figure 2.—A representation of the Arabidopsis genome based on the primary location of each sequenced 5. napus RFLP marker on the Arabidopsis pseudochro-mosomes (megabase distances are indicated to the right of the chromosomes). Duplicate marker locations are indicated in parentheses. Blocks of markers found to be genetically linked in 5. napus are indicated by shading and capital letters (A-F). In the majority of cases, C4B is conserved as a complete block, but in two instances, on N4 and N14, a small section of the block was observed and is represented by C4B'. >° h 776 I. A. P. Parkin et al. N6/N17: The lower half of N6 shows homology to sections of Arabidopsis C5 and C3. The region from block C5B to the bottom of N6 is homeologous but inverted with respect to N17. Two markers on N6/N17 AtC4 PR116\r=v -. pR4ľ^B \ AtC5 es5147\ CA120Ai—■ pW109pW1160v_ CAS* pW200 pN3 H36821 Z33873 = J T756B2-' pN121' pR64 CA144(2)I CA130 p0111 pW153 ATTS2524CA110 CA4 pN102 1E06 CONST ANS' pW155(2)pW189l pOIGO pN91pN105 CA40 N96493 pW212 pNISO 1001 IB06 IHM CA30 RB9998 pW152 pR86 IA09(3) CA69 «49» CA72 pN64 pW167 pO120 pN1D1 p0113 pN202 pW122(2) IA05\ Z18443|2| R30624KX T2080e0* _ CA137^V — pW103\^; pW21BIB12IB08(2)pW135 pW233 T43968^ p0112\ ID11(2) Z18443K p0169 pN184 ATTS2094 pN86K IC01(2)pR3K pN20-. pW176-. pR20- pW154- pR34- CA131% IGD7N H36913-- p0127^ p017(2)- p0168^ pR115v pN63p0155 IC04h pW240- '■-^ "\ 15 >C >D -2cr\ >E < >F 300 250 200 150 100 50 0 H l!rinnnnnn„n ^ ^ ^ ^ rtf ^ ^ j@> ^ rSy ^ ^ ,&> & # # ŕ M.s linkage group N19with both Arabidopsis chromosome 1 and chromosome 5, highlighting the difficulty in identifying the most related Arabidopsis region where there are ancient duplications in the model genome. However, the organization of the different duplicated copies of each block varied with respect to each other, either by the presence of additional rearrangements (see description for Nl/Nll above) or by the number of comparative markers [see description for N3(N17)/ N13 above]. In Arabidopsis, 81% of the comparative loci positioned on the genome mapped to conserved regions present in at least six copies within the B. napus genome (Table 2). Eighty-six percent of the mapped length of the B. napus genome, which was arranged in conserved blocks, was found in at least six copies (Table 2). These results corroborate previous suggestions based on more limited data that the Brassica diploid genomes have evolved through a hexaploid ancestor. However, the presence of seven copies of some Arabidopsis regions within the B. napus genome suggests that further segmental duplication events may have occurred subsequent to any polyploidy event(s). Consequences of duplication within the Arabidopsis genome: The majority of the conserved Arabidopsis blocks, including those known to be part of duplicated regions within Arabidopsis, are each found between five and seven times within the B. napus genome. Effectively, this means that the duplicated regions of the Arabidopsis genome are found between 10 and 14 times within the B. napus genome; similarly, recent physical mapping carried out in B. napus identified 12 regions within the B. napus genome homologous to a small duplicated region of the Arabidopsis genome (Rana et al. 2004). These data suggest that the large segmental genomic duplications found within Arabidopsis occurred in the common ancestor of the two lineages prior to the formation of a Brassica hexaploid ancestor. These data are also consistent with the fact that the last round of genome duplication is believed to have occurred in Arabidopsis between 65 and 90 million years ago (Lynch and Conery 2000; Simillion et al. 2002; Raes et al. 2003) whereas the separation of the Arabidopsis and Brassica lineages is dated somewhere between 12 and 24 million years ago (Koch et al. 2000). Since the divergence of these two species one would expect the independent loss of redundant duplicate genes from both species. Several such losses from the Arabidopsis genome were observed. For example, on Nl and Nil, the upper parts of the linkage groups are colinear with the long arm of Arabidopsis chromosome 4 (Figure 1). Nonetheless, a number of Brassica loci were identified by probes (IC06, CA87, pN52, pN67) originating from Arabidopsis chromosome 2. Although these probes were found in regions identified as being duplicated between chromosomes 2 and 4 of Arabidopsis (http://www.tigr.org/tdb/e2kl/athl/Arabidopsis_ genome_duplication.shtml), they showed no homology to the Arabidopsis chromosome 4 sequence. Thus, Brassica has maintained duplicate copies of these sequences within the region equivalent to chromosome 4, whereas Arabidopsis has lost them. In some instances the duplications evident within the Arabidopsis genome have made it difficult to identify the most similar region shared between the two species. For example, loci on B. napus linkage group N19 show strong homology to both chromosome 5 block C and the duplicated region on Arabidopsis chromosome 1 block D (Figure 4). Conservation of chromosome landmarks between the two species: The position of each Brassica centromere has yet to be accurately determined relative to the genetic linkage maps. However, RFLP mapping of artifactual telocentric chromosomes in Brassica aneu-ploids placed the centromere of linkage group N12 between markers pW177E3 and p05b, the centromere of group N13 between pW181a and pN96b, and the centromere of group N14 between markers pN151b and pW130a (Kelly 1996). Additionally, integration of the cytogenetic and genetic linkage maps of B. oleracea positioned the centromere of linkage group Arabidopsis/5. napus Comparative Map 779 Ol (equivalent to Nil) between markers pN152El and p0168El (Howell et al. 2002). In the proposed centromeric region of N12, four coincident markers were mapped with homology to Arabidopsis sequences that span the centromere on chromosome 4, suggesting conservation of chromosome position between the species. It is possible that with sufficient marker data the Arabidopsis centromeric positions could be used to predict functional and ancestral centromeric regions in Brassica chromosomes. The latter would arise since a hexaploid derived from a lower chromosome progenitor, which likely had between 5 and 8 chromosomes, originally would have had between 15 and 24 functional centromeres, which were then reduced to 10 and 9 in the Brassica A and C genomes, respectively. As in the case of N12, there were a number of instances where the density of markers across the Arabidopsis centromere was insufficient to identify a conserved block in B. napus. However, the loci identified by these same markers were tightly linked in B. napus, and in the case of Nl 1, N12, and N13 there was further cytological evidence suggesting the centromere location. Each of these putative centromeric regions is indicated in Figure 1. As evidenced by numerous small segments of colinearity flanking these provisional centromeric regions on Nil, N12, and N14, it appears that the neighboring regions are prone to rearrangements and evolve rapidly compared to more distal regions. The karyotype of B. oleracea indicates that linkage group 07 (equivalent to N17) is an acrocentric chromosome and has a strongly hybridizing 45S locus at the terminus of the short arm (Howell et al. 2002). This region of N17 shows homology to the short arm of Arabidopsis chromosome 2 and coincidently one of the two nucleolar organizer regions (NORs) of Arabidopsis also maps to the terminus of the short arm of chromosome 2 (Franz et al. 1998). DISCUSSION In this study, by allowing minor disruptions in conserved regions it was possible to identify 21 conserved blocks within Arabidopsis, which could be replicated and rearranged to cover almost 90% of the mapped length of B. napus. A minimum number of 74 gross rearrangements, with 38 in the A genome and 36 in the C genome, can be estimated to have separated the two lineages since their divergence 14—24 MYA (Koch et al. 2000). This lies between two previously published figures derived from Brassica/Arabidopsis comparative mapping: 19 chromosomal rearrangements separating B. oleracea from Arabidopsis (Lan et al. 2000) and 90 separating B. nigra from Arabidopsis (Lagercrantz 1998). Detecting rearrangements is influenced by a number of variables including the number and type of available comparative markers, the level of polymorphism within a mapping population, and the method of determining colinearity between species. For Lan et al. (2000) the lower figure was probably due to a low density of comparative markers and for Lagercrantz et al. (1998) the much higher figure was due in part to the approach used to identify syntenous regions, with no allowance made for minor disruptions of colinearity, and was exacerbated by the inclusion of markers thought to be single copy in Arabidopsis but now known to be multi-copy Comparing estimates of the level of rearrangements in lineages is problematic because of the inherent difficulties in comparing data sets and due to variation in the estimated divergence times. With that proviso, considering the data presented here, the level of rearrangement observed in the Brassiceae tribe, as represented by the A and C genomes of B. napus, is relatively high when compared with related species from the Brassicaceae family. Recently, the genetic maps of Capsella rubella (Lepideae tribe) and Arabidopsis lyrata (Sisymbrieae tribe) have been compared to the sequence map of A. thaliana (Boivin et al. 2004; Kuittinen et al. 2004). On the basis of the comparison to the A. thaliana genome, analysis of the two maps indicates equivalent linkage group organization, with the eight chromosomes of C. rubella, A-H, aligning with the A. lyrata chromosomes, AL1-AL8, respectively. This demonstrates that both species evolved from a common ancestor. A. lyrata and C. rubella are estimated to have diverged from Arabidopsis 5 and 10 MYA, respectively (Boivin et al. 2004; Kuittinen et al. 2004). A limited number of major chromosomal rearrangements, ^6—13, separate these two species from A. thaliana. In addition, no major rearrangements have separated A. lyrata from C. rubella. Although it is not possible to align all the conserved blocks identified in this study with the C. rubella and A. lyrata genomes, the junctions of a number of the rearrangements identified between these two species and A. thalianacorrespond to the ends of conserved blocks identified in this study. However, none of the chromosomal rearrangements that separate A. lyrata and C. rubella from A. thaliana appear to be common to the Brassiceae lineage. The fact that the majority of the identified conserved segments are found in at least six copies in B. napus and that 81% of the comparative loci, which define the conserved blocks in Arabidopsis, are mapped to these triplicated regions, is consistent with a proposed hexaploid ancestor for the diploid Brassica progenitor. However, it could still be argued that the observed pattern of duplicated segments is the result of several smaller independent segmental duplications following a single whole-genome duplication event, a mode of evolution that would require a significant number of independent duplication events. Polyploidy has been a prevalent mechanism of evolution within the angiosperms and it has been estimated that 30-70% of species have undergone at least one round of chromosome doubling during their evolutionary development (reviewed in Wendel 2000). There is also well-documented evidence 780 I. A. P. Parkin et al. for extensive chromosomal rearrangements in newly resynthesized Brassica polyploids (Parkin et al. 1995; Song et al. 1995). Thus genome triplication followed by a small number of insertions/deletions/translocations would provide the simplest explanation for the present structure of the Brassica diploid genome. In this study, the overall picture is one of conservation ofgene content and gene order between the genomes of Arabidopsis and B. napus. The average length of the conserved blocks identified between the two species was 14.8 cM in B. napus and 4.8 Mb in Arabidopsis. However, for at least seven B. napus linkage groups, half their mapped length was equivalent to one conserved region of the Arabidopsis genome. Undoubtedly, the Brassica genomes have undergone restructuring during their evolution from a common ancestor of Arabidopsis, but this has not prevented the maintenance of large stretches of similarity, in some cases equivalent to 9 Mb of contiguous Arabidopsis genomic sequence. In a number of instances, the comparative mapping provisionally suggests correspondence of centromere positions between the two species. The large conserved regions found across the different genomes, punctuated by numerous smaller blocks of similarity, suggest that there are preferential regions for chromosome breakage and subsequent rearrangements. The publication of the genome sequence of Arabidopsis has opened up many avenues of research with the expectation that these endeavors would have applications in the study of the more complex genomes of crop plants (Arabidopsis Genome Initiative 2000). The complete sequence allowed the resolution of the exact physical positions for ^30,000 genes, 50% of which have no known function and any of which could hold the key to understanding a number of important agronomic traits. The comparative map suggests that the model genome of Arabidopsis can be widely exploited to infer the genetic basis of traits in its economically valuable Brassica crop relatives. In the identified conserved regions, the Arabidopsis genomic sequence should be an excellent resource for identifying useful markers, targeting the genie regions, since they show on average 86% sequence identity. Accurately mapping the genes controlling target phenotypes in large segregating Brassica populations should allow candidate genes to be inferred from the Arabidopsis sequence. However, due to the duplicated nature of the Brassica genomes it will be difficult to predict whether any particular Arabidopsis gene will have been maintained in all the duplicate copies. Comparative genomic sequencing in other plant species suggest that there almost certainly will have been numerous rearrangements at the level of microsynteny (Bennetzen and Ramakrishna 2002). Limited physical mapping in B. oleracea identified only one potential inversion and one gene in a nonsyntenic position; however, there was obvious interspersed gene loss from the different triplicated regions (O'Neill and Bancroft 2000). In addition, recent physical mapping in the B. napus genome uncovered a similarly small number of disruptions in the microsynteny but evidence of changes in gene content between the homologous Brassica segments compared to the homologous Arabidopsis regions (Rana et al. 2004). Genomic sequence data of such regions from Brassica species will allow the extent to which the duplicate copies have been conserved to be determined, provide insights into the mechanism underlying the rearrangements differentiating the different copies, and allow an estimate of the relative age of the different duplication events. We thank Cambridge Plant Breeders, Twyfords, and Advanta who supported the development of the Brassica RFLP probes (pN, pO, pR) and the construction of the genetic linkage maps of B. napus described in Parkin et al. and in Sharpe et al. 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